File S1 - Interplay of the Bacterial Ribosomal A-Site, S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

Supporting files. Figure S1, Flipping of A1492 and A1493 (without the antibiotic) during MD simulations. a) The pseudo-dihedral angle used to describe flipping (here shown for A1492) defined by the four pseudoatoms: CM1 – centre of mass of the neighboring base pair (C1409 and G1491), CM2 – centre of mass of the ribose of the neighboring nucleotide (G1491), CM3 – centre of mass of the flipping nucleotide ribose (A1492), CM4 – centre of mass of the flipping nucleobase (A1492). The values of for A1492 and A1493 versus time in the variants: b) WT, c) K42A and d) R53A in each MD trajectory (the simulation numbers are shown in parentheses in [b]). in the variants: b) WT, c) K42A and d) R53A in each MD trajectory (the simulation numbers are shown in parentheses in [b]). The typical direction of A1492 and A1493 flipping movement during mRNA decoding process (through the minor groove of the rRNA helix) is indicated by arrows in panel (b). Figure S2, Convergence of MD simulations for the wild-type systems with and without paromomycin. a) Evolution of RMSD from the starting structures for the solute heavy atoms in each of the 3 simulations in WT and WT variants. b)-d) Cumulative RMS fluctuations. The average fluctuations were calculated for the selected subsets of residues as a function of time: b) the h44 helix, c) ‘head’ of the S12 protein and d) the H69 helix. Panel (e) shows the membership of mutual A1492 and A1493 conformations in time to different clusters in three WT simulations (without paromomycin). Different colors mark different clusters. Clustering was done for conformations gathered from all 3 trajectories, with 3.5 Å radius (see ‘Methods’ in the main text). Figure S3, Structural stability in the simulated systems. a) RMSD (P/C) versus time for h44 and H69 helices and the “tail” of the S12 protein in the chosen MD simulations. The structures with the highest RMSD in the trajectories selected from those presented in (a): b) h44 and c) S12 and the three nucleotides mentioned in the text: U911 – in light green, C912 – blue, A913 – red; red arrow indicates the direction of conformational change from the initial structure. Figure S4, Distributions of sugar pucker phase for A1492 and A1493 in MD simulations. The regions corresponding to C2′- endo and C3′- endo configurations are shaded. The data are cumulative from three independent MD trajectories per simulation variant. Figure S5, Sugar pucker phase of A1492 vs A1493 gathered from 170 crystal structures of the bacterial ribosome. The C2′- endo and C3′- endo ranges are indicated. Figure S6, Structural stability of paromomycin in MD simulations: a) RMSD in time for the ring IV of paromomycin (see Fig 1e, main text) in the two simulations: one of WT with stable PAR and the other of R53A variant, with exceptionally unstable antibiotic. b) Conformational change of paromomycin in the R53A trajectory: the initial structure – in yellow, the conformation with the highest RMSD – colored by atom type. Figure S7, Positions of A1492 vs A1493 in crystal structures of the bacterial ribosomes. The plots of C1′(C1409)-C1′(G1491)-C1′(A1492)-N9(A1492) versus C1′(C1407)-C1′(G1494)-C1′(A1493)-N9(A1493) dihedral angles. These angles were shown to characterize well flipping of the nucleotides in bulges [84] and they are analogical to the angles defined in Fig S1a in File S1. The values close to 0 correspond with the flipped-in conformations, while the values close to 180 – the flipped-out states. The arrows indicate the two directions of flipping-out: through the minor and major groove (marked by continuous and dotted lines, respectively). Data are gathered from 167 structures. Figure S8, Comparison of H69 conformations in different crystal structures of bacterial ribosomes. Abbreviations: E. c. – E. coli, D. r. – D. radiodurans. PDB codes are given. Sections S1–S6, Describe the force field considerations (Section S1), dynamics and stability of the S12 protein (Section S2), overall mobility of the h44 and H69 helices (Section S3), fluctuations of A1492 and A1493 (Section S4), conformations of paromomycin (Section S5), and how the antibiotic bound in the A-site affects the conformational dynamics of A1492 and A1493 (Section S6). Table S1, Deviations from the initial configuration during MD for the chosen subsystems: averages of RMSD ( standard deviation, in Å), calculated for P and C atoms with respect to the initial structure. For each simulation variant the data from 3 MD trajectories are averaged over time. Table S2, Duration of H-bonds created between N3 nitrogen of K43 and h44 nucleotides (expressed as % of trajectory time). For each MD simulation run it is calculated as an average of occupancies for H-bonds of K43(N3) with 13 hydrogen acceptors: G1491 (O1P, O3′), A1492 (O1P, O2P, O2′, O4′, O5′, N3, N7), A1493 (O1P, O2P, N1, N7). The data are also averaged over 3 trajectories for each variant (standard deviations in parentheses). One WT simulation is excluded from averaging (as in this case the K43⋅⋅⋅⋅h44 interactions are not formed) and it is treated as an exception. Table S3, Percentage of time when H-bond between C1412(O2′) and C910(O2′) atoms is formed during MD simulations (data are from three trajectories for each variant).
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